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Title: A metabolic pathway for catabolizing levulinic acid in bacteria

Microorganisms can catabolize a wide range of organic compounds and therefore have the potential to perform many industrially relevant bioconversions. One barrier to realizing the potential of biorefining strategies lies in our incomplete knowledge of metabolic pathways, including those that can be used to assimilate naturally abundant or easily generated feedstocks. For instance, levulinic acid (LA) is a carbon source that is readily obtainable as a dehydration product of lignocellulosic biomass and can serve as the sole carbon source for some bacteria. Yet, the genetics and structure of LA catabolism have remained unknown. Here, we report the identification and characterization of a seven-gene operon that enables LA catabolism in Pseudomonas putida KT2440. When the pathway was reconstituted with purified proteins, we observed the formation of four acyl-CoA intermediates, including a unique 4-phosphovaleryl-CoA and the previously observed 3-hydroxyvaleryl-CoA product. Using adaptive evolution, we obtained a mutant of Escherichia coli LS5218 with functional deletions of fadE and atoC that was capable of robust growth on LA when it expressed the five enzymes from the P. putida operon. Here, this discovery will enable more efficient use of biomass hydrolysates and metabolic engineering to develop bioconversions using LA as a feedstock.
Authors:
 [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [2] ;  [2] ;  [2] ;  [2] ;  [3] ;  [2] ;  [1] ; ORCiD logo [1]
  1. Univ. of Wisconsin, Madison, WI (United States)
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
Publication Date:
Grant/Contract Number:
AC02-05CH11231
Type:
Accepted Manuscript
Journal Name:
Nature Microbiology
Additional Journal Information:
Journal Volume: 2; Journal ID: ISSN 2058-5276
Publisher:
Nature Publishing Group
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES
OSTI Identifier:
1399002

Rand, Jacqueline M., Pisithkul, Tippapha, Clark, Ryan L., Thiede, Joshua M., Mehrer, Christopher R., Agnew, Daniel E., Campbell, Candace E., Markley, Andrew L., Price, Morgan N., Ray, Jayashree, Wetmore, Kelly M., Suh, Yumi, Arkin, Adam P., Deutschbauer, Adam M., Amador-Noguez, Daniel, and Pfleger, Brian F.. A metabolic pathway for catabolizing levulinic acid in bacteria. United States: N. p., Web. doi:10.1038/s41564-017-0028-z.
Rand, Jacqueline M., Pisithkul, Tippapha, Clark, Ryan L., Thiede, Joshua M., Mehrer, Christopher R., Agnew, Daniel E., Campbell, Candace E., Markley, Andrew L., Price, Morgan N., Ray, Jayashree, Wetmore, Kelly M., Suh, Yumi, Arkin, Adam P., Deutschbauer, Adam M., Amador-Noguez, Daniel, & Pfleger, Brian F.. A metabolic pathway for catabolizing levulinic acid in bacteria. United States. doi:10.1038/s41564-017-0028-z.
Rand, Jacqueline M., Pisithkul, Tippapha, Clark, Ryan L., Thiede, Joshua M., Mehrer, Christopher R., Agnew, Daniel E., Campbell, Candace E., Markley, Andrew L., Price, Morgan N., Ray, Jayashree, Wetmore, Kelly M., Suh, Yumi, Arkin, Adam P., Deutschbauer, Adam M., Amador-Noguez, Daniel, and Pfleger, Brian F.. 2017. "A metabolic pathway for catabolizing levulinic acid in bacteria". United States. doi:10.1038/s41564-017-0028-z. https://www.osti.gov/servlets/purl/1399002.
@article{osti_1399002,
title = {A metabolic pathway for catabolizing levulinic acid in bacteria},
author = {Rand, Jacqueline M. and Pisithkul, Tippapha and Clark, Ryan L. and Thiede, Joshua M. and Mehrer, Christopher R. and Agnew, Daniel E. and Campbell, Candace E. and Markley, Andrew L. and Price, Morgan N. and Ray, Jayashree and Wetmore, Kelly M. and Suh, Yumi and Arkin, Adam P. and Deutschbauer, Adam M. and Amador-Noguez, Daniel and Pfleger, Brian F.},
abstractNote = {Microorganisms can catabolize a wide range of organic compounds and therefore have the potential to perform many industrially relevant bioconversions. One barrier to realizing the potential of biorefining strategies lies in our incomplete knowledge of metabolic pathways, including those that can be used to assimilate naturally abundant or easily generated feedstocks. For instance, levulinic acid (LA) is a carbon source that is readily obtainable as a dehydration product of lignocellulosic biomass and can serve as the sole carbon source for some bacteria. Yet, the genetics and structure of LA catabolism have remained unknown. Here, we report the identification and characterization of a seven-gene operon that enables LA catabolism in Pseudomonas putida KT2440. When the pathway was reconstituted with purified proteins, we observed the formation of four acyl-CoA intermediates, including a unique 4-phosphovaleryl-CoA and the previously observed 3-hydroxyvaleryl-CoA product. Using adaptive evolution, we obtained a mutant of Escherichia coli LS5218 with functional deletions of fadE and atoC that was capable of robust growth on LA when it expressed the five enzymes from the P. putida operon. Here, this discovery will enable more efficient use of biomass hydrolysates and metabolic engineering to develop bioconversions using LA as a feedstock.},
doi = {10.1038/s41564-017-0028-z},
journal = {Nature Microbiology},
number = ,
volume = 2,
place = {United States},
year = {2017},
month = {9}
}

Works referenced in this record:

MicrobesOnline: an integrated portal for comparative and functional genomics
journal, November 2009
  • Dehal, P. S.; Joachimiak, M. P.; Price, M. N.
  • Nucleic Acids Research, Vol. 38, Issue suppl_1, p. D396-D400
  • DOI: 10.1093/nar/gkp919

Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression
journal, February 2013

The Pseudomonas putida Crc global regulator controls the hierarchical assimilation of amino acids in a complete medium: Evidence from proteomic and genomic analyses
journal, June 2009
  • Moreno, Renata; Martínez-Gomariz, Montserrat; Yuste, Luis
  • PROTEOMICS, Vol. 9, Issue 11, p. 2910-2928
  • DOI: 10.1002/pmic.200800918

Rapid Quantification of Mutant Fitness in Diverse Bacteria by Sequencing Randomly Bar-Coded Transposons
journal, May 2015
  • Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.
  • mBio, Vol. 6, Issue 3, Article No. e00306-15
  • DOI: 10.1128/mBio.00306-15

Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009
  • Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
  • Nature Methods, Vol. 6, Issue 5, p. 343-345
  • DOI: 10.1038/nmeth.1318

Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System
journal, January 2015
  • Jiang, Yu; Chen, Biao; Duan, Chunlan
  • Applied and Environmental Microbiology, Vol. 81, Issue 7, p. 2506-2514
  • DOI: 10.1128/AEM.04023-14

Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing
journal, September 2015

Basic local alignment search tool
journal, October 1990
  • Altschul, Stephen F.; Gish, Warren; Miller, Webb
  • Journal of Molecular Biology, Vol. 215, Issue 3, p. 403-410
  • DOI: 10.1016/S0022-2836(05)80360-2

Production of levulinic acid and use as a platform chemical for derived products
journal, February 2000
  • Bozell, Joseph J.; Moens, L.; Elliott, D. C.
  • Resources, Conservation and Recycling, Vol. 28, Issue 3-4, p. 227-239
  • DOI: 10.1016/S0921-3449(99)00047-6

The binding of propionyl-CoA and carboxymethyl-CoA to Escherichia coli citrate synthase
journal, July 1995
  • Man, Wai-Jin; Li, Yan; David O'Connor, C.
  • Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, Vol. 1250, Issue 1, p. 69-75
  • DOI: 10.1016/0167-4838(95)00044-U

RNA-guided editing of bacterial genomes using CRISPR-Cas systems
journal, January 2013
  • Jiang, Wenyan; Bikard, David; Cox, David
  • Nature Biotechnology, Vol. 31, Issue 3, p. 233-239
  • DOI: 10.1038/nbt.2508